Zdzisław Kleinrok

Limbic seizures produced by pilocarpine in rats: Behavioural, electroencephalographic and neuropathological study

Behavioural, electroencephalographic and neuropathological responses to increasing doses of pilocarpine (100-400 mg/kg) administered intraperitoneally to rats were studied. At the dose of 400 mg/kg pilocarpine produced a sequence of behavioural alterations including staring spells, olfactory and gustatory automatisms and motor limbic seizures that developed over 1-2 h and built up progressively into limbic status epilepticus. Smaller doses showed different threshold for these behavioural phenomena but a similar time course of development. The earliest electrographic alterations occurred in the hippocampus and then epileptiform activity propagated to amygdala and cortex. Subsequently electrographic seizures appeared in both limbic and cortical leads. The ictal periods recurred each 5-15 min and were followed by variable periods of depression of the electrographic activity. The sequence of electrographic changes correlated well with the development of behavioural phenomena. Histological examination of frontal forebrain sections revealed disseminated, apparently seizure-mediated pattern of brain damage. Neuropathological alterations were observed in the olfactory cortex, amygdaloid complex, thalamus, neocortex, hippocampal formation and substantia nigra. Pretreatment of animals with scopolamine (20 mg/kg) and diazepam (10 mg/kg) prevented the development of convulsive activity and brain damage. These results show that systemic pilocarpine in rats selectively elaborates epileptiform activity in the limbic structures accompanied by motor limbic seizures, limbic status epilepticus and widespread brain damage. It is suggested that a causative relationship between excessive stimulation of cholinergic receptors in the brain and epileptic brain damage may exist.

Effects of calcium channel inhibitors upon the efficacy of common antiepileptic drugs.

Diltiazem and nifedipine (both 1.25 mg/kg) markedly potentiated the protective action of carbamazepine and diphenylhydantoin against maximal electroshock-induced seizures in mice. These calcium channel inhibitors retained their activity at lower doses. Diltiazem and nifedipine (2.5 mg/kg) also moderately potentiated the efficacy of phenobarbital and valproate. Verapamil (up to 10 mg/kg) was not effective against the action carbamazepine, diphenylhydantoin, phenobarbital, and valproate. None of the calcium channel inhibitors used (up to 40 mg/kg) influenced aminophylline-induced convulsions and mortality. Moreover, the anti-aminophylline activity of valproate and phenobarbital was not potentiated by the calcium channel inhibitors in doses up to 10 mg/kg. Further, combination of carbamazepine, ethosuximide, and trimethadione with the calcium channel inhibitors (up to 10 mg/kg) did not offer any protection against aminophylline-induced convulsions. It can be concluded that calcium channel inhibitors enhance the protective efficacy of some antiepileptics against electroconvulsions. A pharmacokinetic interaction does not seem to be responsible for this effect.

Influence of calcium channel inhibitors upon the anticonvulsant efficacy of common antiepileptics against pentylenetetrazol-induced convulsions in mice

Among three calcium channel inhibitors studied, nifedipine (20 mg/kg) moderately inhibited pentylenetetrazol (115 mg/kg, s.c.)-induced convulsions, whilst diltiazem (up to 20 mg/kg) and verapamil (up to 20 mg/kg) were without effect. The combinations of nifedipine (10 and 20 mg/kg) with valproate (100 mg/kg) or phenobarbital (6.25 mg/kg) resulted in significant protection against pentylenetetrazol-induced seizures. Combined treatment of nifedipine (5-20 mg/kg) with ethosuximide (100 mg/kg) also provided a clearcut anticonvulsant action. The antiepileptic drugs alone, in the above doses, were ineffective. The combination of diltiazem (10-20 mg/kg) and ethosuximide (100 mg/kg) produced protection against pentylenetetrazol, comparable to that of ethosuximide (200 mg/kg) alone. No pharmacokinetic interactions were found in the case of ethosuximide, whilst nifedipine (10 mg/kg) increased the levels of phenobarbital and valproate in plasma. The combination of diltiazem with the remaining antiepileptics were ineffective. Verapamil (up to 20 mg/kg) was without effect upon the action of the antiepileptic drugs tested. Finally, none of the calcium channel inhibitors studied influenced the action of diazepam (0.2 mg/kg). It may be concluded that combinations of ethosuximide, with either nifedipine or diltiazem, may be promising for the treatment of absence epilepsy.

NG-Nitro-L-arginine differentially affects glutamate- or kainate- induced seizures

The effects of nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (NNA) on seizures induced by excitatory amino acids, bicuculline, pentylenetetrazol and pilocarpine were studied in mice. NNA (10 and 40 mg kg-1, i.p.) enhanced the susceptibility to intracerebroventricular (i.c.v.) kainate (KA) which was reflected by a decrease in its convulsant dose 50% (CD50) from 0.66 nmol to 0.38 and 0.29 nmol/mouse, respectively. Also, NNA (40 mg kg-1) increased the KA-induced mortality. Conversely, NNA (40 mg kg-1) produced an anticonvulsant effect against i.c.v. glutamate whose CD50 value was significantly elevated from 0.49 mumol to 0.84 mumol/mouse. The convulsive activity of i.c.v. N-methyl-D-aspartic acid (NDMA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) was not affected by the pretreatment with NNA (40 mg kg-1). NNA (5-40 mg kg-1) also potentiated the convulsive action of systematic KA and KA-induced mortality but (up to 40 mg kg-1) remained without effect on seizures produced by bicuculline, N-methyl-D, L-aspartic acid (NMDLA), pentylenetetrazol, and pilocarpine. Only bicuculline-produced lethality was significantly enhanced. It may be concluded that the manipulation of the NO level affects differently seizures arising from a diffuse stimulation of glutamate receptors and seizures resulting from an activation of an individual subtype of these receptors. It is noteworthy that in the majority of convulsive tests used in this study, NNA exerted no modulatory effect.

Influence of melatonin upon the protective action of conventional anti-epileptic drugs against maximal electroshock in mice

Melatonin (50 mg/kg; 60 min before the test) significantly raised the electroconvulsive threshold in mice. The protective action of melatonin (50 mg/kg) in the electroconvulsive threshold test was reversed by aminophylline, picrotoxin and bicuculline. Melatonin at the subconvulsive dose of 25 mg/kg potentiated the anticonvulsive activity of carbamazepine and phenobarbital (ED50 values were significantly decreased from 12.1 to 8.3 and from 18.9 to 11.8 mg/kg, respectively). No potentiation was observed in the case of valproate and diphenylhydantoin (their ED50s were changed from 253 to 249 and from 10.3 to 9.7 mg/kg, respectively). Melatonin did not influence the plasma or brain levels of anti-epileptics studied, so a pharmacokinetic interaction is not probable. Melatonin (25 mg/kg) alone and its combinations with carbamazepine or phenobarbital, providing a 50% protection against maximal electroshock, were devoid of significant motor adverse effects, but caused strong long-term memory deficit. Consequently, it does not seem to be a good candidate for the treatment of epilepsy.

2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline enhances the protective activity of common antiepileptic drugs against maximal electroshock-induced seizures in mice

NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline), a novel and selective AMPA antagonist, was tested to evaluate its influence upon anticonvulsant activity of common antiepileptic drugs in mice. NBQX (10, 20, 40 mg/kg, i.p.) had no influence upon the threshold for electroconvulsions. NBQX (10 mg/kg) enhanced the activity of anticonvulsant drugs decreasing their ED50S against maximal electroshock from 321 to 190 mg/kg for valproate, from 19.5 to 14.5 mg/kg for carbamazepine, from 31.0 to 21.4 mg/kg for phenobarbital, from 17.8 to 9.5 mg/kg for diphenylhydantoin and from 19.5 to 10.5 mg/kg for diazepam. In addition, NBQX (10 mg/kg) failed to impair motor performance and long-term memory determined in the chimney test and passive avoidance task. The combinations of NBQX (10 mg/kg) and carbamazepine, diphenylhydantoin or phenobarbital resulted in no adverse effects. Diazepam (10.5 mg/kg) alone impaired the motor performance and long-term memory and so it did when combined with NBQX. Also retention of the passive avoidance task and motor performance were impaired by valproate alone or given together with NBQX. Finally, NBQX (10 mg/kg) did not affect the plasma level of any antiepileptic drug. It is concluded that non-NMDA glutamate receptor blockade results in the considerable enhancement of the efficacy of common antiepileptic drugs.

The non-competitive AMPA/kainate receptor antagonist, GYKI 52466, potentiates the anticonvulsant activity of conventional antiepileptics

1-(4-Aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), up to 5 mg/kg, did not influence the electroconvulsive threshold but potentiated the anticonvulsant activity of valproate, carbamazepine and diphenylhydantoin against maximal electroshock-induced convulsions in mice. No potentiation was observed in the case of phenobarbital. Moreover, this non-NMDA receptor antagonist did not influence the plasma levels of the antiepileptic drugs studied, so a pharmacokinetic interaction, in terms of total and free plasma levels, is not probable. The combined treatment of GYKI 52466 with either carbamazepine or diphenylhydantoin (providing a 50% protection against maximal electroshock) was devoid of significant side effects (motor and long-term memory impairment). Valproate applied at a dose equal to its ED50 caused serious worsening of motor coordination and long-term memory. It is noteworthy that the combined treatment of GYKI 52466 with valproate was superior to valproate alone, as regards adverse effects. The results suggest that concomitant administration of GYKI 52466 with some conventional antiepileptic drugs may offer a novel approach in the treatment of epilepsy.

LY 300164, a novel antagonist of AMPA/kainate receptors, potentiates the anticonvulsive activity of antiepileptic drugs.

LY 300164 [7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4, 5H)-2,3-benzodiazepine], administered intraperitoneally up to 2 mg/kg, did not influence the threshold for electroconvulsions. In doses of 2.5-4 mg/kg, LY 300164 significantly raised the threshold. In subprotective doses against electroconvulsions, this excitatory amino acid receptor antagonist enhanced the protective activity of intraperitoneally given valproate, carbamazepine and diphenylhydantoin against maximal electroshock-induced convulsions in mice. The anticonvulsive action of phenobarbital was potentiated by LY 300164 only at 2 mg/kg. The non-N-methyl-D-aspartate receptor antagonist did not affect the plasma levels of the antiepileptic drugs, so a pharmacokinetic interaction is not probable. Combined treatment with LY 300164 (2 mg/kg) and the antiepileptics studied (providing 50% protection against maximal electroshock) did not impair the motor performance of mice, evaluated in the chimney test. Valproate, at its ED50 of 280 mg/kg against maximal electroshock, produced motor impairment. As shown in the passive avoidance task, combination of LY 300164 (2 mg/kg) with valproate or diphenylhydantoin resulted in impairment of long-term memory. Alone among the antiepileptics, valproate (280 mg/kg) and phenobarbital (28.5 mg/kg) disturbed long-term memory. The results suggest that blockade of glutamate-mediated events via non-NMDA receptors leads to enhancement of the anticonvulsive activity of conventional antiepileptics. Some combinations of LY 300164 with antiepileptic drugs were superior to these antiepileptics alone in terms of their lack of adverse effects.

Mitochondrial toxin 3-nitropropionic acid evokes seizures in mice

3-Nitropropionic acid, a potent inhibitor of succinate dehydrogenase which thus compromises cellular energy metabolism, evoked convulsions in mice in a dose-dependent manner. CD50 for clonic seizures was 158.5 (144.1-174.3) mg/kg. Tonic seizures were not observed. Broad-spectrum anticonvulsants, namely diazepam, phenobarbital and valproate, prevented the occurrence of 3-nitropropionic acid-induced seizures with ED50 of 4.9 (3.1-7.6), 33.1 (17.9-61.0) and 389.7 (351.2-432.3) mg/kg, respectively. Diphenylhydantoin-like drugs (diphenylhydantoin, and carbamazepine), anti-absence drugs (trimethadione and ethosuximide) and acetazolamide were ineffective. The characteristics of 3-nitropropionic acid-induced seizures resembled those of convulsions evoked by another mitochondrial toxin, aminooxyacetic acid.

Impairment of brain kynurenic acid production by glutamate metabotropic receptor agonists.

THE role of glutamatergic mechanisms in kynurenic acid (KYNA) production was evaluated in vitro. The selective ionotropic agonists NMDA, kainate and AMPA did not affect KYNA synthesis. Agonists of metabotropic (mGLU) and ionotropic receptors: quisqualate, L-glutamate and L-aspartate as well as agonists of mGLU receptors: (±)−1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) diminished KYNA production with different potency. None of the studied mGLU antagonists such as (S)-4-carboxyphenylglycine, α-ethylglutamic acid or (RS)-α-methylserine-O-phosphate affected the basic or L-glutamate-inhibited synthesis of KYNA. It might be hypothesized that the impairment of KYNA production following the application of mGLU receptor agonists is related to their effects exerted upon the novel subtype of mGLU receptor.